| A genetically modified organism (GMO) is defined as an organism whose genetic material has been altered by the insertion of a modified gene or a gene from another organism using molecular biology technologies, so that the shape, nutritional quality and/or consumption quality will change to meet the need of human beings. When using the GMO as direct food or as a raw material for the production of food it is called "genetically modified food (GMF)"With the development of genetic engineering technologies, GMF appears in response to the needs of the time, and comes into the life of people with amazing speed. In2011, the planting area of genetically modified crops was up to160million hectares,94times more than the1.7million hectares in1996and the kinds of GMF have amounted to a great number. GMF has brought rich food supply and a huge economic efficiency to humans, but so far, the safety of GMF for human health has been queried. It has been reported that some GMF can cause harm to humans such as poisoning, allergic reactions, reduction of the ability to resist to viruses and changes in the flora balance of the intestinal tract. In order to ensure the consumers right to know and choose, many countries and regions all over the world have issued the food industry to compulsory or voluntary label their products for GMF. In2002, the department of agriculture of China issued "agriculture genetically modified organisms identification management method", requiring that5types of17kinds of GMF products must be marked.The marking of the GMF is based on the detection of the genetically modified ingredients. Therefore it is very important to build a set of accurate, fast and efficient detection technologies for the detection of genetically modified ingredients, and the high quality of GMF DNA templates gives a promising starting point for genetic testing.The isolation of nucleic acids consists of two steps, extraction and purification. Extraction frees the nucleic acids from the cell and purification separates the nucleic acids from cellular components such as proteins, polysaccharides, salt and other impurities. The extraction methods includes a chemical extraction method (surfactant SDS, CTAB, high salt etc.), an enzyme extraction method (proteinase K, lysozyme repressible enzyme, etc.) and a mechanical extraction method (grinding, etc.). The most commonly used purification method is organic solvent extraction and precipitation. Another one is purification by a medium which uses a solid phase purification medium that separates the DNA from other substances by using the characteristics of selective adsorption in particular conditions.It is very difficult to extract DNA from GMF due to the complex ingredients of food and serious degradation of DNA during food processing. At present many domestic and foreign scholars actively explore and study all kinds of efficient GMF DNA extraction methods. According to the GMF processing degree, a set of DNA extraction methods was constructed and optimized for all the processing levels of food used in this study.First of all, for genetically modified agricultural products, we used a Chelex-100method to extract DNA from transgenic farm products rapidly. Chelex-100is a chemical ion chelating resin composed by styrene and divinylbenzene and it can lead to cell membrane rupture. The DNA was released from the nucleus in an alkaline environment (pH10～11) at100℃. At the same time Chelex-100can combine with many non-nucleic acid materials that may affect the next step of the analysis. Because Chelex-100can eliminate the non-nucleic acid organic matter effectively and prevent DNA from degradation through combining with metal ions, it is economically, convenient and efficient. At present it is already widely used to extract forensic traces like blood, tissue, semen, bones and so on.In this study,the national standard (GB/T19495.3-2004) CTAB-1method and the Chelex-100method have been compared to extract the DNA of genetically modified soya GTS40-3-2. The statistical analysis of the DNA concentration and purity indicated that the Chelex-100was of higher concentration (t=10.424, P=0.000), and was of lower purity (t=12.684. P=0.000). However as a template for PCR amplification there was no obvious difference between the two methods. We also did a study on the stability of the DNA, extracted from different agricultural products by the two different methods, when saved at-20℃. The result showed that there was no obvious difference between the two methods. The Chelex-100method was also comfirmed a quick method to isolate DNA from other genetically modified farm products.Secondly, for mild-to-moderately processed food, we used the modified traditional CTAB method. This is a classical way to extract plant DNA and also the most reported method in the literature to extract DNA of GMF. However the traditional CTAB method was slightly modified because the traditional method is tedious and therefore time-consuming, and uses complex reagent compositions such as phenol and chloroform which can easily cause environmental pollution problems. This study improved the traditional CTAB method by using a silicon film adsorption column to purify DNA, thereby reducing the use of toxic organic solvents such as poisonous phenols and greatly shortening the operation time. The national standard (GB/T19495.3-2004) CTAB-1method and the improved CTAB method have been compared for the extraction of DNA from genetically modified soya GTS40-3-2. The statistical analysis of DNA concentration and purity indicated that the improved CTAB method was of higher concentration and purity (for concentration t=9.471, P e=0.001; for purity t=3.253, P=0.031), which is significantly different, showing that the method is ideal. It was also a good method for DNA extraction from mild-to-moderately processed food.Thirdly, because the amount of DNA isolated from oil is low and degraded more seriously after processing, this research made use of the silicon film adsorption column as medium, combined with precipitation agent salmon DNA sperm, which established a stable and effective DNA extraction method from edible oil. Its application in5brands of11kinds of edible oil extraction of DNA, the effect of PCR, LAMP,real time fluorescence quantitative PCR detection were ideal.Finally, high quality DNA template is a promising starting point for the detection of GMF, however genetically modified ingredients detection is the ultimate goal. Transgenic food testing includes protein detection based on exogenous protein targets and nucleic acid detection. The actual protein detection is little work, however, costs are high, and the operation time is long and after processing of GMF,.most of the protein denaturation which makes the detection rate low. The testing of nucleic acid levels includes PCR technology, gene chip technology, etc. Simple PCR detection technology is prone to contamination and false positives are easily produced. The gene chip technology is a new technology in the life science field and is fast, sensitive, and accurate, has high throughput and can be used to identify and screen a large number of samples. Target gene marker genes in a microarray experiment process is an important link.Multiplex PCR amplification of marker is to join the Cy3-dCTP fluorescence labeling in multiplex PCR amplification process, and it is high specificity.Multiplex PCR combined with gene chip analysis can complement each other, using the advantages of the two different technologies. The PCR amplification of marker method combined with the chip fluorescence probe hybrid technology will achieve high sensitivity and specificity. Based on5kinds of13varieties of GMF’s genetic screening genes, we designed specific primers and probes and established an oligonucleotide microarray detection method based on a multiplex PCR for5kinds of13varieties of GMF, thereby achieving the ideal test results.Through experimental study, we reached the following results:1. An economically, convenient and quick DNA extraction method was designed, based on the Chelex-100method, which was suitable for high throughput screening and identification of genetically modified agricultural products.2. The traditional CTAB method was improved by shortening of the operation time by using silicon film adsorption columns to purify DNA. The application of DNA extraction in mild-to-moderately processed food obtained the ideal effect.3. A good, versatile, cost-effective oil DNA extraction method was designed to efficiently extract stable DNA from cooking oil.4. Various probes and samples have been introduced as quality controls during the process of the hybridization, which eliminated the false positive/negative results while not harming the microarray performance.5. We used multiplex PCR method for genome amplification marker, completed laboratory test, the experimental results show that the multiplex PCR amplification labeling technique is a good DNA marker technology;6. We designed26oligonucleotide probes according to5varieties13strains of GMF, prepared a GMF food gene chip.On various strains of standard products were detected, results and standards in line with the rate of100%, indicating that the chip can be applied in the screening and detection of transgenic samples.In summary, the DNA extraction and purification of GMF is the basis and prerequisite of GMF detection and it is very important to establish a perfect DNA extraction method. A GMF examination methods are highly specific, have high efficiency and stable results according to the different processing degrees of GMF. The DNA can satisfy the requirements of qualitative PCR, real-time fluorescence quantitative PCR, the LAMP and gene chip testing and other molecular biology experiments. GMF Oligo microarray detection method based on the multiplex PCR can more effectively improve the specificity and sensitivity, through comprehensively using the high efficiency and high throughput of the multiplex PCR amplification marker and the highly sensitive and highly specific characteristics of gene chips. Some small scale experimental results of the GMF standard substance application show that the specificity of the gene chips in this study is good, the effect is stable, and the platform is reliable. We can use this method to detect different GMF synchronously, rapidly and accurately. It provides a quick, supervision sensitive, accurate testing method for screening and can detect a large amount of GMF. |
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